HIV-1 Proviral DNA Qualitative Detection by PCR, Blood
Method Description Describes how the test is performed and provides a method-specific reference
HIV-1 Proviral DNA is extracted and purified from whole blood by the automated MagNA Pure LC instrument (Roche Applied Science, Indianapolis, IN) using the MagNA Pure LC DNA Isolation Kit-Large Volume. The specimen input volume is 500 mcL with a final elution volume of 110 mcL.
The double-stranded DNA is denatured by heat to expose the target region to labeled primers. The amplification target, a highly conserved region of the GAG genome, is bordered by the primer pair SK145 and SKCC1B. The 2 biotinylated oligonucleotide primers complementary to the amplification target sequence will bind to the target region on the DNA (native or internal control DNA). rTth polymerase links deoxynucleotide triphosphates, (dATP, dGTP, dCTP, dUTP) extending in the 5' to 3' direction to produce biotinylated complementary DNA sequences called amplicons. During PCR, controlled fluctuations in temperature allow repeated denaturation, annealing, and extension processes resulting in a geometric increase in the target sequences. DNA copies from previous cycles become templates in subsequent amplification periods. The resulting amplicon is 155 bp in length. HIV-1 internal control is added to identify clinical specimens containing inhibitory substances or test failure due to inadequate individual specimen processing. The internal control is introduced into each specimen during the extraction steps to serve as an extraction and amplification control for each independently processed specimen.
Uracil-N-glycosylase (UNG) is used to prevent contamination from previous PCR reactions. UNG will excise any dUTP found in previously amplified DNA. (Naturally occurring DNA will contain dTTP.) Before the PCR reaction is initiated, the thermal cycler is set at 55 degrees C to optimally activate UNG for 2 minutes. After the amplification cycles are completed, the UNG is inactivated by a denaturing solution containing sodium hydroxide.
The amplified DNA is incubated in polystyrene wells containing immobilized bovine serum albumin-conjugated probe (SK 102) that is specific to the biotinylated amplicons. After incubating for 1 hour, the unbound reactants are washed away. An avidin-horseradish peroxidase conjugate is added and incubated. Unbound reactants are washed away. A substrate solution containing hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine (TMB) is added. In the presence of hydrogen peroxide, the bound horseradish peroxidase catalyzes the oxidation of TMB to form a color complex. The reaction is stopped by the addition of a weak acid and the absorbance is read at 450 nm. A fixed optical density cutoff is used to determine whether a specimen is positive or negative.(Package insert: Amplicor HIV-1 DNA Test, version 1.5, Roche Diagnostics, Indianapolis, IN, March 2005)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Analytic Time Defines the amount of time it takes the laboratory to setup and perform the test. This is defined in number of days. The shortest interval of time expressed is "same day/1 day," which means the results may be available the same day that the sample is received in the testing laboratory. One day means results are available 1 day after the sample is received in the laboratory.
Maximum Laboratory Time Defines the maximum time from specimen receipt at Mayo Medical Laboratories until the release of the test result
Specimen Retention Time Outlines the length of time after testing that a specimen is kept in the laboratory before it is discarded
Performing Laboratory Location The location of the laboratory that performs the test